In recent years it has become evident that basic differences exist between pituitary cellular regulation in primates and that in rodents. I propose to establish dispersed pituitary cells from macaques in stable monolayer cultulre to directly examine the cellular regulation of prolactin secretion. The pituitary cells will be plated on an extracellular matrix secreted by bovine corneal endothelial cells. The prolactin secretion of the cells will be monitored by radio immunoassay, and the amount of newly synthesized prolactin will be determined with 3-H leucine incorporation and immunoprecipitation. Various culture media will be used to define the optimum conditions for maintenance of stable hormone production without fibroblast overgrowth. Experiments will be executed to characterize the sensitivity and secretory dynamics of the lactotropes to neural substances that may regulate prolactin secretion, such as dopamine, thyrotropin-releasing hormone, and vasoactive intestinal polyeptide. Each factor will be tested in a dose-response fashion to obtain a median effective dose and thus an indication of its biological potency. The specificity and stereoselectivity of each factor will be verified by competition experiments with specific antagonists when possible. In addition, the responsiveness of the cells will be characterized in the presence and absence of estradiol-17 Beta since this steroid modifies prolactin secretion, but discrepancies have been reported between rodents and primates. The in vitro results will be validated with in vivo experiments and will be correlated with binding assay results. Because of the difficulty and cost of obtaining primate tissue, I further propose to establish a primate prolactin-secreting cell line after transformation with simian virus 40. The clones obtained from transformation must be examined for hormone production and receptor integrity. Ultimately, the effects of neural and peripheral factors on receptor dynamics (receptor nu;mbers, receptor turnover, and internalization of ligand-receptor complexes) and on the intracellular synthesis and packaging of prolactin wil be studied. Finally, I propose to examine the feasibility of cocultulring catecholamine-producing cells and prolactin-secreting cells for further study on the dopaminergic regulation of prolactin secretion.